Pakistan J. Zool., vol. 52(2), pp 447-455, 2020.
DOI: https://dx.doi.org/10.17582/journal.pjz/20180718100758
In vitro Assessment and Characterization of the
Growth and Life Cycle of Leishmania tropica
Qaisar Jamal*, Akram Shah, Syed Basit Rasheed and Muhammad Adnan
Department of Zoology, University of Peshawar, Peshawar, Pakistan
ABSTRACT
Present study aimed at determining the in vitro life cycle of Leishmania tropica. Temporal variability in
the morphology and various morphotypes of the promastigotes were noted. Acquisition and propagation
of axenic amastigotes was assessed. During promastigote culture, log, mid-log, and late-log phases
were observed respectively on day 4, 5 and 6. The stationary phase was observed on day 7. The day
following inoculation, most of the promastigotes were nectomonads having long and slender bodies with
roughly uniform width from anterior to near posterior end, having as long flagella as one and a half
to twice the body. The nectomonads changed to leptomonads with a ratio of 5% and 44% on day 2
and 3 respectively. The body to flagellum ratio decreased in leptomonads giving a wider and shorter
appearance than the nectomonads. On day 4, the log phase, the metacyclic promastigotes appeared in
the culture. The metacyclic to leptomonad to nectomonad ratio was 27%, 43% and 29% respectively.
The ratio of metacyclics steadily increased towards the mid-log (53%), late-log (67%), and stationary
phase (83%). Metacyclics had long flagella and short stumpy cell bodies with anterior end much broader
than the posterior as compared to that in leptomonads and nectomonads. The posterior end of the body
was bearing a slender tail like extension . During logarithmic growth, 17 % of promastigotes were found
dividing. During a 10 days transformation of axenic amastigotes, the day following inoculation to the day
3 different morphological forms were observed. The day 7 onward only rounded no flagellum (RNF=4147%) and oval no flagellum (ONF= 53-59%) forms were present. Viability remained 96-98% during
transformation. Bulk growth of the promastigotes for prolong (over a week) duration resulted in the
acidification of medium and change to viable amastigotes that could be successfully retransformed to
promastigotes. Use of orthophosphoric acid for acquiring acidic medium to transform promastigotes to
amastigotes proved effective.
INTRODUCTION
L
eishmaniasis, widely distributed in tropics and neotropics, is a vector-borne protozoal disease that ranks
9th among the infectious diseases regarding global disease
burden (Reithinger et al., 2007; Berry and BerrangFord, 2016). Human and mammalian leishmaniasis is
characterized by a myriad of clinical spectra ranging
from localized and ulcerative skin lesions (cutaneous
leishmaniasis) to non-ulcerative nodules (diffused
cutaneous leishmaniasis) to the inflammation of mucus
membranes (muco-cutaneous leishmaniasis) to infection of
visceral organs (visceral leishmaniasis; the fatal form of the
disease causing maximum deaths). Execept in some cases,
where one species can infect different foci, the different
forms are caused by different species (Reithinger et al.,
2007). It is currently reported from 98 countries around
the globe with an overall global prevalence of 12 million
cases, annual death incidence of 70,000 and 350 million
*
Corresponding author: qaisar.jamal21@uop.edu.pk
0030-9923/2020/0002-0447 $ 9.00/0
Copyright 2020 Zoological Society of Pakistan
Article Information
Received 18 July 2018
Revised 11 May 2019
Accepted 03 June 2019
Available online 16 January 2020
Authors’ Contribution
QJ planned the work, did the
experimental lab work and wrote the
primary manuscript. AS supervised
the work. SBR helped in data analysis,
MA helped in prepration of the
manuscript.
Key words
Leishmania, Promastigotes,
Axenic amastigotes, Morphology,
Characterization
people at risk. The cutaneous and visceral leishmaniases
have an annual global incidence of 0.7-1.2 million and
0.2-0.4 million respectively. In Pakistan it is estimated that
approimately 21-35 thousand cases of CL occur annually
(Jamal et al., 2015; Reithinger et al., 2007; Rodrigues et al.,
2015; Rock et al., 2015). Most of the VL cases (90%) are
reported from India, Brazil, Sudan, Bangladesh, Ethiopia
and southern Sudan having annual death toll of 20-40
thousands (Gradoni, 2013; Kaur et al., 2015). Colombia,
Peru, Brazil, Costa Rica, Algeria, North Sudan, Ethiopia,
Afghanistan, Iran, and Syria contribute 70-75% of the 10
million cases of the cutaneous leishmaniasis (Ambit et al.,
2011; Alvar et al., 2012).
Leishmania, the etiologic agent of leishmaniasis,
possesses a heteroxenous life cycle, swinging between
flagellated promastigote and aflagellated amastigote form
occurring respectively in invertebrate (sandfly gut) and
vertebrate (a variety of lizards and mammals including
human) macrophages. Both the forms of the parasite can be
maintained in axenic cultures using defined media (CysneFinkelstein et al., 1998; Sacks, 1989; Sacks et al., 2000).
The promastigote form has been demonstrated to show
morphologically and functionally different morphotypes
Q. Jamal et al.
448
both in vivo (Killick-Kendrick, 1979; Sacks and Perkins,
1985) and in vitro (Sacks et al., 1995; Lira et al., 1998).
As compared to in vivo existence of morphotypes (Schlein
et al., 1993; Killick-Kendrick et al., 1996; Gossage et
al., 2003; Oliveira et al., 2009; Ramalho-Ortigao et al.,
2010; Dostálova and volf, 2012), in vitro existence is less
established. Limited number of studies, as stated before,
with few species of Leishmania, have investigated these
morphological forms in axenic development. Evaluation of
such variability is really important for infection kinetics of
the parasite and for their conversion to axenic amastigotes.
Amongst the variety of different morphotypes, metacyclic
form is capable of causing infection both in vivo and in
vitro. In axenic culture, the metacyclogenesis reaches at
different rates for different species. The published record
for L. tropica metacyclogenesis in axenic growth is scarce.
This study aimed at elucidation of metacyclogenses
of L. tropica promastigotes and acquisition of axenic
amastigotes.
MATERIALS AND METHODS
Growth of promastigotes
Promastigotes were cultured in RPMI-1640 medium
(Sigma Life Sciences) at a density of 1× 106/mL in 25 mL
nonvented culture flask. The medium was supplemented
with 10 % heat inactivated fetal calf serum (hiFCS, Sigma
Life Sciences), 100 µg/mL streptomycin (Sigma Life
Science), 100 IU penicillin (Sigma Life Sciences) and
L-glutamine. The growth was monitored daily by counting
in neubauer haemocytometer over a week.
Evaluation of morphological variations of promastigotes
during culture
Morphologic variability was monitored daily through
Giemsa stained smear over a period of one week. Usually
a drop of culture was smeared on an albuminized slide and
fixed in methanol after air drying at room temperature.
Various trial concentrations of the aqueous methanol
were tried to fix the parasite cells. Fixed smears were
stained with Giemsa stain. Smears were observed under
the oil immersion objective and photographed. Temporal
variation in the length, width, and shape of the cell and
length of the flagellum were qualitatively assessed to
determine different promastigote morphotypes in the
axenic culture. Metacyclics were also revealed through
scanning electron microscopy.
Cultivation of axenic amastigotes and evaluation of
morphology
Axenically grown amastigotes have become a great
deal of interest for drug screening and biochemical as
well as molecular variability between the promastigote
and amastigote stages. A variety of different protocols
have been followed to axenically transform promastigotes
to amastigotes for different species. In the present study,
1x106/ mL promastigotes in the stationary phase were
grown at 37⁰C in humidified conditions lacking CO2 in
RPMI 1640 medium fortified with 20 % heat inactivated
fetal calf serum (hiFCS: Sigma Lifescience), 100 µg/mL
streptomycin, 100 IU penicillin and L-glutamine. The pH
of the medium was adjusted at 4.4 using phosphoric acid
(85% aqueous solution BDH Chemicals). Giemsa staining,
and phase contrast microscopy was used to monitor the
transformation on daily bases while fully transformed
amastigotes were also visualized through scanning
electron microscope.
During the promastigote to amastigote transformation,
the change in cell shape was observed daily by Giemsa
smearing and expressed in terms of percentage from
elongated promastigote form in the initial inoculum to the
fully round form with a variety of oval forms in between.
Presence and absence and length of the flagellum was also
noted. Reduction in the cell size was determined through
micrometry. The size measurement was mostly based
on the fully round and oval individuals. The size was
expressed as mean of 20 randomly selected individual cell
measurements.
Acquisition of axenic amastigotes by starvation
Amastigotes were acquired in the in vitro culture
when the culture was kept starved without medium change,
accidentally for the first time, and then several times when
repeated intentionally.
Viability during transformation
Viability of cells is a necessary aspect in the drug
sensitivity and biological studies. The viability of
leishmanial cells during promastigote to amastigote
transformation was checked daily over a week using trypan
blue exclusion and was expressed as percentage of viable
cells. After complete transformation, the amastigotes were
culture in the normal RPMI-1640 as mentioned above for
promastigotes to check the viability.
RESULTS
Growth curve of L. tropica KWH23
The growth of L. tropica KWH23 was observed by
growth curve over one week period of the culture (Fig.
1). For the first three days of the culture, a stunted growth
was seen. On day 4, the culture reached log phase of the
growth. On day 5 to 6 the mid and late log phases of L.
tropica promastigotes development were achieved. On
449
Growth and Life Cycle of Leishmania tropica
day 7, the promastigote culture entered the stationary
phase (1×107/mL). In stationary phase, the promastigotes
became sluggish and were slithering around. Cells were
looking stumpy with long flagella. The body looked like
a carrot with bulging anterior and tapering posterior ends.
Fig. 1. Growth curve of Leishmania tropica KWH23 over
a week.
Revision of strain nomenclature
Leishmania tropica KWH23 strain was originally
isolated from a 5-year-old boy belonging to Jamrud (an
endemic focus for cutaneous leishmaniasis) area of Kyber
Agency, then known as Federally Administered Tribal
Area (FATA) Pakistan in 2010 visiting Kuwait Teaching
Hospital, Peshawar, Khyber Pakhtunkhwa Pakistan for
intra lesional treatment. It was isolated by Nazma Habib
Khan during her Ph D from London School of Hygiene
and Tropical Medicine, London UK. Since then it has been
labelled with the name where KWH comes from the name
of the hospital where the strain originated and 23 refers to
the serial number of the patient studied. According to the
strain nomenclature for Leishmania isolates (leishnet.net),
it must contain four parts separated by a slash (/). The first
part is a reference to the host from which it is isolated,
usually a four-letter abbreviation; first of which represents
the class of the host in the hierarchic classification followed
by the first three letters of the genus of the host. The second
part is a two-letter abbreviation of the country of origin, a
list of which is available online. The third part is the year
of isolation and the fourth part is not fixed and is on the
choice of the person who has done the isolation, which
might be the original patient ID in the hospital record
or of the researcher, thus revising this strain to MHOM/
PK/2010/KWH23.
Axenic promastigote morphotypes
Various morphotypes were determined through one
week development. Culture was initiated from stationary
phase promastigotes. It was infinite passage number of the
strain KWH23. On day 1st almost all of the promastigotes
449
were nectomonads (Fig. 2B) having long, slender and
progressively tapering bodies with long flagella about
one and a half to twice as long as the body. Next day
only 5% of the promastigotes were leptomonads (Fig.
2C) with still slender and long cells and longer flagella
almost indistinguishable from those of nectomonads.
In leptomonads, the cell body becomes somewhat less
tapering posteriorly and cell to flagellum ratio decreses.
On day third, the ratio of leptomonads rose to 44 %.
During the log phase on day 4, metacyclics appeared in the
culture. The matacyclics to leptomonads to nectomonads
were 27 %, 43 % and 29 % respectively. Metacyclics had
long flagella and short stumpy cell bodies with anterior
end much broader than the posterior as compared to that
in leptomonads and nectomonads. The posterior end of the
body was bearing a slender tail like extension (Fig. 2 D, E,
F). Cell number got increased till day 6 ( late log phase)
where onward the stationary phase reached and the number
of metacyclics increased progressively. Microscopic
assessment of the in vitro development of L. tropica in the
present study represented few procyclic (Fig. 2A).
Fig. 2. Different stages of L. Tropica KWH23 promastigotes
in culture (See text for description).
Division of L. tropica promastiogtes in axenic development
During logarithmic growth, 17 % of promastigotes
were found dividing, so features of the dividing cells were
observed (Fig. 3). Most of the individuals duplicated the
flagellum first following division of the kinetoplast and
the nucleus respectively. However, in rare cases, division
of the kinetoplast and nucleus preceded flagellar splitting
(Fig. 3C).
Cultivation of axenic amastigotes
After 24 hours of incubation under the abovementioned conditions, the metacyclic promastigotes started
to change in the overall morphology. As per previously
described criteria of transformation from promastigote to
amastigote, major changes have been observed regarding
Q. Jamal et al.
450
Table I. Promastigote to amastigote transformation ratio over 10-day span based on cell morphology.
% Shape →
Day ↓
RNF RBVF
RSF
RLF
ONF
OBVF
OSF
OLF
ENF
EBVF
ESF
ELF
1.
03
05
05
02
04
25
27
09
08
05
04
03
2.
10
09
5
01
23
27
20
02
0
0
02
01
3.
09
08
07
01
31
40
03
01
0
0
0
0
4.
19
13
0
0
68
0
0
0
0
0
0
0
5.
31
06
0
0
63
0
0
0
0
0
0
0
6.
29
09
0
0
62
0
0
0
0
0
0
0
7.
41
0
0
0
59
0
0
0
0
0
0
0
8.
44
0
0
0
56
0
0
0
0
0
0
0
9.
47
0
0
0
53
0
0
0
0
0
0
0
10.
43
0
0
0
57
0
0
0
0
0
0
0
RNF, rounded no flagellum; RBVF, rounded barely visible flagellum; RSF, rounded stumpy flagellum; RLF, rounded long flagellum; ONF, oval no flagellum; OBVF, oval barely visible flagellum; OSF, oval stumpy flagellum; OLF, oval long flagellum; ENF, elongated no flagellum; EBVF, elongated barely
visible flagellum; ESF, elongated stumpy flagellum; ELF, elongated long flagellum.
Fig. 3. Division of the L. tropica promastigotes in axenic
development. Cell with 2 flagella single kinetoplast
and single nucleus (A), Cells with single flagellum, two
kinetoplasts and single nucleus (B), Cell with Single
flagellum, two kinetoplasts and two nuclei (C) and Cells
with two flagella, two kinetoplasts and two nuclei near
to the completion of cytokinesis (D). Solid arrows show
kinetoplasts and the others indicate flagella.
inoculation, all the different morphotypes were seen. Up
till day 3 a mixture of the morphotypes were present. Day
4 and onward only the rounded no flagellum and oval no
flagellum along with few percent of rounded barely visible
flagellum forms were present in the culture. Although
the transformation process started right on the following
day of inoculation, but proper amastigote forms were
transformed after day 4 and onward. The fully transformed
culture contained the rounded and oval forms. However,
oval cells predominated the rounded ones throughout
the culture. Our recent observations of amastigotes of L.
tropica isolated from CL lesions from the type locality of
the strain showed both oval and round forms (Fig. 4).
the morphological changes in cell shape and length of
flagellum (Fig. 4).
Shape and flagellar changes during transformation
The changes in shape of the cell and length of the
flagellum during promastigote to amastigote transformation
has been noted (Table I). The results were presented as
percentage per 100 cells counted in a Giemsa stained smear.
In a 10 days daily monitoring of the transformation process
several different morphotypes were observed. There was a
gradual loss of the flagellum with individual variability.
As is clear from the table on day 1; the following day of
Fig. 4. Promastigote to amastigote transformation.
Change in the cell morphology and loss of flagellum is
seen progressively from day 0-10. Lesion amastigotes are
indicated by the arrow in the last inset.
Cell size as well as length of flagellum experienced
tremendous reduction during the transformation of
Growth and Life Cycle of Leishmania tropica
451
amastigotes. This reduction in cell size was abrupt. There
was a maximum reduction in cell length on day first
and second after which the size of the amastigote form
became rather more constant (Table II). Fully transformed
amastigote form varied from little oval or near round to
fully rounded (Table III).
Table II. Transformation of promastigotes to
amastigotes in terms cell size reduction over a week.
Days
0
Mean Variance Stdev.
CI Var.
10.96667 2.363529 1.537377 1.4-5.4
1
4.511111 0.704575 0.83939
2
3.927778 0.296242 0.544281 0.17-0.7
0.4-1.7
CI Stdev.
1.2-2.1
1.18-1.45
0.413-0.84
3
3.277778 0.065359 0.255655 0.036-0.15
0.189-0.387
4
3.111111 0.045752 0.213896 0.025-0.1
0.158-0.316
5
3.194444 0.062908 0.250816 0.035-0.138 0.187-0.371
6
3.111111 0.045752 0.213896 0.025-0.1
7
3.138889 0.053105 0.230444 0.030-0.118 0.173-0.343
0.158-0.316
Table III. Size of the fully round and oval form of
amastigote.
Fully Round Form
Mean
Variance Stdev.
CI var,
CI Stdev
Day-5 3.18
0.101714 0.318927 0.05-0.25
Day-10 3.146667
0.095524 0.309069 0.048-0.225 0.219-0.474
0.224-0.5
Oval form
Length 3.653333
0.132667 0.364234 0.071-0.33 0.266-0.574
Width
0.084
2.94
0.289828 0.045-0.21 0.212-0.458
Viability during transformation
Viability of cells is a necessary aspect in the drug
sensitivity and biological studies. The viability of
leishmanial cells during promastigote to amastigote
transformation was checked daily during the study
period using trypan blue exclusion and was expressed as
percentage of viable cells. It varied from 96-98%.
Natural acquisition of axenic amastigotes
During the study period, when several times the
culture was kept starved for a week to ten days at 26⁰C
and absence of CO2 the promastigotes changed to
amastigotes due to change in pH of medium although this
occurrence was purely accidental for the first time. Colour
of the medium changed from light pink to light yellow
with final pH of 4.8-5. The culture medium (having 10
% FCS) started to change its colour after day 3 with pH
falling to 6.8 having only promastigotes. On day 5th the
451
pH fell to 6 but it still was having only promastigotes in
late log phase. The pH fell gradually to 5.5 on day 7 with
82% promastigotes and the rest were partially changed
amastigotes having rounded to oval or stumpy body but
long flagellum. On day 9 the pH came to about 5 and the
percentage of amastigotes increased to 80% mostly having
rounded body and stumpy to rudimentary flagellum. On
day 10 and onward the pH came to 4.8 and the ratio of
amastigotes rose to 91%. Viability of the amastigotes was
97 % as evaluated by trypan blue exclusion. The culture had
1.2 x 107 amastigotes per mL. When grown in medium for
promastigotes such amastigotes successfully transformed
back to promastigotes. Natural transformation was slower
than the one performed in the defined medium for axenic
amastigotes. However, the results were satisfactory, and
the yield was enough. Such transformation yielded enough
axenic amastigotes for one-time use. The amastigotes
could be successfully propagated through sub-passaging
in the defined medium.
Some requirements for counting, fixation and smearing of
Leishmania tropica KWH23
Counting is a necessary process in culturing and
propagation as well as in vitro and in vivo anti-leishmanial
assays of compounds. Inactivation of the L. tropica
promastigote is needed for counting in haemocytometer
as they are actively motile. Amastigotes are, however,
stationary and need no such inactivation. During the present
study, 10-25 % ethanol was found far beyond satisfactory
to inactivate them to be easily studied under microscope in
haemocytometer. Ten percent aqueous methanol solution
for 10-30 seconds gave excellent results in fixing both
the promastigote and amastigote forms of the parasite. To
attach the parasite to the slide surface, egg albumin was
used. Fresh albumin was less efficient in adhesion of the
parasite to slide but a day to a week older albumin was
found very much effective in attaching the cells to the slide
surface. Amastigotes were found less efficient in sticking
to the surface of albumenized slide than the promastigotes.
A 2 % V/V working Giemsa stain for 10-20 minutes at
room temperature gave excellent results in staining both
the promastigotes and amastigotes. Such a working
Giemsa stain worked satisfactorily for 2 weeks. Fresh stain
replacement after two weeks is, however, recommended.
DISCUSSION
Morphological variability of promastigote stage in
different species of Leishmania have been described in the
vector host. Several different morphotypes occurs during
development in sand fly. Each of these morphotypes also
presents bichemical variability and varied virulence to
452
Q. Jamal et al.
the host (Schlein, 1993; Ismael et al., 1998; Gossage et
al., 2003; Kamhavi, 2006; Ramalho-Ortigao et al., 2010;
Oliveira et al., 2009; Dougall et al., 2011). The description
of such morphotypes in axenic development is apparently
difficult, partly because of temporal association of such
developmental form and partly due to great deal of speciesspecific variations. For these reasons, limited number of
studies have been attempted to assess the morphological
forms in the axenic cultivation of the promastigotes in
few Leishmania species, L. donovani, L. mexicana and
L. major being the prominent ones (da Silva and Sacks,
1987; Sacks, 1989). However, axenic growth of the
promastigotes reflect several different morphotypes seen
in the in vivo development in the vector host. Different
morphotypes reported in the sand fly vector can also be
detected in the in vitro culture (Sacks et al., 1984; Gossage
et al., 2003; Lei et al., 2010a)
Initiated from lesion amastigotes, Bates (1994)
demonstrated, various morphotypes of L. mexicana
axencally. The initial amastigote to promastigote
transformation was achieved in 2 days. Logarithmic
growth, with a mix of different morphotypes in the
multiplicative stage, was noted to occur on days 3 and
4. At this stage (3-4 days), few infective metacyclic
promastigotes were observed. Most of the promastigotes
on the late day 3 were longest (15.22 ± 3.49 μM) and
extremely slender than noted on any other day and were
carrying long flagella. However, flagellar and body length
were comparable. These were morphologically very much
similar to the nectomonads noted in in vivo studies. On day
7th , the promastigotes reached to the stationary phase and
metacyclogenesis was on the peak. In 9 days old axenic
culture, however, the density of metayclics reached to
the 95 %. The metacyclic promastigotes became stumpy,
broader anteriorly, needle sharp posteriorly and were with
long flagella. The flagella were 1-2 times longer than the
cell size (7.59 ± 1.15 μM). These findings are in a good
consensus with what we have come across in this study.
Short and slender metacyclic promastigotes with long
flagella have been demonstrated in stationary cultures by
other investigators also (Sacks and Perkinn, 1984; Grimm
et al., 1991).
The ratio of metacyclics to other forms is in inverse
relationship to the number of passages. It usually decreases
in long-term axenic cultivation. Fresh cultures that have
gone through few passages get fast metacyclogenesis with
higher densities of metacyclics than the older cultures
(Cysne-Finkelstein et al., 1998). Results of the present
study are strongly supported by the in vivo results of Dougall
et al. (2011). Their study have reported similar kind of
morphological forms of promastigote of a yet unidentified
species of Leishmania isolated from day biting midges
of genus Forcipomyia. They found very long slender
nectomonads with long flagella. The leptomonads they
isolated were rather short banana like with relatively short
flagella. Their metacyclics, however, varied from needle
long with long flagella to anteriorly broad and posteriorly
needle sharp having long flagella. Lei et al. (2010)
have reported the existence of procyclic, nectomonad,
leptomonad and metacyclics in culture. They have shown
very similar body lenghts for procyclics, leptomonads
and metacyclics (6-11 μM). However, nectomanads were
found more than 12 μM in length. All of the stages they
noted have been of very similar morphology to what we
have found in the present study.
Lectins obtained from several different plants
including peanut, is used to purify metacyclic promastigotes
from culture. This compound agglutinates the procyclic
and other forms leaving metacyclics. Metacyclics and
non-metacyclics separated from stationary culture of L.
amazonensis have been reported to be morphologically
different. The non-metacyclics shown so have been long
and almost equally broad along their length except near
the posterior tip. These have been shown to have very long
flagella. These are characteristics of the nectomonads. The
mectacyclics have been short and stumpy with broader
anterior half and much narrower posterior half. These have
been shown to possess very long flagella (Saravia et al.,
2005). This work support our results in the present study.
Soares et al. (2005) also noted similar kind of results for
L. braziliensis. The procyclic form was found to be very
much variable in size of the cell a flagellum. They probably
considered all the non-metacyclic forms as procyclic that
varied in cell size from rounded to elongated individuals.
However, the metacyclics were defined as smaller and
thinner,usually posteriorly, bodies with long flagella.
When grown under different conditions of
temperature and pH, the metacyclic promastigotes of L.
mexicana showed different growth and pro-amastigote
transformation kinetics. At a temperature of 26° C and pH
of 7.2 no transformation occurred. With these conditions,
however, in some experiments transitional forms have
been seen for short duration. At 26° C and pH 5.5, greater
degree of transformation have been observed but it was
followed by reversion to promastigotes in 24 hours.
When the transformation was checked at 32° C and pH
7.2, within 12 hours almost all of the cells entered to the
intermediate forms that sustained for 48 hours. Although
the intermediate forms did not revert to the promastigote
stage but also did not fully transformed to amastigote
stage. A temperature and pH panel of 32° C and 5.5 was
the most effective in bringing the desired transformation
over 48-96 hours (Bee et al., 2001). These conditions for
transformation are in close coincidence of our observation.
Growth and Life Cycle of Leishmania tropica
453
Similarly, the observation of various transitional stages
in the mentioned study is shared with what we noted in
this study. However, we did not see any reversion like the
mentioned workers at the ideal combination they used. A
difference from what they have noted that we achieved
quite good amastigote transformation at 26° C and 4.8 pH
in the natural transformation.
Metacyclic promastigotes of L. mexicana grown
in Schnider’s medium suppliemented with 20% fetal
calf serum at 25° C and 5.5 pH have been successfully
transformed to amastigotes by rising simply the temperature
to 32° C in the parent culture. After 24 hours of the changed
conditions of the temperature, more than 80% of the cells
changed to rounded amastigotes with complete loss of
flagellum. The remaining were the intermediate forms
differing in the cell and flagellar morphology. By the day
2, however, 100% transformation was seen (Bates, 1994).
These findings present a strong correlation of temperature
and pH to the transformation process. Species-specific
variability can, however, not be ruled out. Generally, a
temperature and pH combination of 32-37° C and 4.5-5.5
seems feasible for bringing satisfactory transformation.
Entirely transformed amastigotes may be ovoid to fully
rounded in almost all species of Leishmania.
CONCLUSION
Morphological variability in the in vitro development
of L. tropica can be determined microscopically. The
present study will provide guidelines for the subsequent
workers in acquisition of any specific morphotype for
their use. This will also help them in acquiring axenic
amastigotes of the species. A comprehensive protocol for
processing the promastigotes as well as amastigotes for
both light and electron microscopy will be of help to the
coming workers.
ACKNOWLEDGEMENTS
The authors are extremely thankful to Professor
Nasim Siddiqui Memorial Parasitology Laboratory and
Leishmania Molecular and Culture Lab of the Department
of Zoology, University of Peshawar for providing material
support for this study.
Statement of conflict of interset
The authors hereby declare no conflict of interest.
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